The present invention relates generally to the discovery of a neuropeptide, designated cortistatin, that shares structural similarity with somatostatin yet, unlike somatostatin, enhances slow-wave sleep. Cortistatin nucleic acid and encoded polypeptides along with anti-cortistatin antibodies are useful in both screening methods, diagnostic methods and therapeutic methods related to modulation of sleep and disorders thereof.
Changes in arousal state from waking. to sleep are accompanied by dramatic changes in the electroencephalogram (EEG). The low amplitude, high frequency pattern of the awake EEG becomes dominated by high amplitude, low frequency synchronized activity in slow-wave sleep (SWS), followed sequentially by rapid eye movement (REM) sleep (Steriade et al., Science, 262:679-685 (1993). Acetylcholine (ACh) plays a key role in the transition of the different phases of sleep (Shiromani et al., Ann. Rev. Pharmacol. Toxicol., 27:137-156 (1987). SWS requires low ACh levels whereas REM sleep is characterized by high ACh content. Also, these phases of sleep have been shown to be differentially sensitive to a number of endogenous neuropeptides and cytokines, including somatostatin, which is known to increase REM sleep without significantly affecting other phases (Borbely et al., Physiol. Rev., 69:605-670 (1989).
The present invention describes the cloning and characterization of cortistatin, a novel neuropeptide that has been discovered to be a sleep-modulating molecule with effects opposing those mediated by somatostatin. Cortistatin, however, exhibits strong structural similarity to somatostatin. Thus obtaining a cDNA clone from screening brain-specific libraries, the mRNA of which clone is translated into a naturally occurring physiologically active protein, is yet a further example of such molecules described in U.S. Pat. Nos. 4,900,811 and 5,242,798.
Although cortistatin has now been determined to be the product of a different gene, because of its structural similarity to somatostatin as well as functional aspects described herein, cortistatin is a new member of the somatostatin family whose distribution is primarily restricted to GABAergic cortical interneurons.
GABAergic neurons have been shown to finely modulate the output of principal neurons of the cerebral cortex and hippocampus (Buhl et al., Nature, 368:823-828 (1994), areas that have been implicated in arousal state and complex cognitive functions, including learning and memory (Wilson et al., Science, 265:676-679 (1994).
The neuropeptide somatostatin was first described as a hypothalamic peptide that inhibited growth hormone release (Brazeau et al., Science, 179:77-79 (1973), and has since been implicated in many physiological phenomena, including hippocampal function and REM sleep generation (Danguir, Brain Res., 367:26-30 (1986). In the hippocampus, somatostatin is present largely in a particular set of interneurons. See, Hendry et al., Proc. Natl. Acad. Sci., USA, 81:6526-6530 (1984); Schemchel et al., Neurosci. Lett., 47:227-232 (1984); and Morrison et al., Brain Res., 262:344-351 (1983). Somatostatin may modulate the output of pyramidal neurons primarily by depressing neuronal excitability, in part via enhancement of the voltage-dependent potassium M current. See, Moore et al., Science, 239:278-280 (1988) and Schweitzer et al., Nature, 346:464-466 (1990). Pharmacological studies have shown that somatostatin also interacts with cholinergic (Araujo et al., J. Neurochem., 55:1546-1555 (1990) and Mancillas et al., Proc. Natl. Acad. Sci., USA, 83:7518-7521 (1986) and GABAergic (Freund et al., Nature, 336:170-173 (1988) systems, among others, thus modulating systems thought to underlie different aspects of behavior.
As shown in the present invention, despite the physical similarities between somatostatin and cortistatin, administration of cortistatin in vivo depresses neuronal electrical activity but, unlike somatostatin, induces low frequency waves in the cerebral cortex and antagonizes the effects of acetylcholine on hippocampal and cortical measures of excitability, thus providing a mechanism for cortical synchronization related to sleep.
A mammalian neuropeptide, designated cortistatin, has now been discovered, cloned, sequenced and characterized for biological activity. Cortistatin is expressed in cortical and hippocampal mammalian interneurons, has an amino acid residue sequence similar to but distinct from somatostatin, and has neurologic properties including neuronal depression, sleep modulation and enhanced slow wave sleep.
The basic discovery of a new polypeptide of this nature provides a variety of embodiments, including compositions, methods of their use, and screening procedures for the identification of additional useful compositions.
In one embodiment, the invention describes a substantially isolated cortistatin protein and a cortistatin polypeptide including an amino acid residue sequence defining a cortistatin polypeptide having a sequence that corresponds to a sequence in the Sequence Listing selected from the group consisting of SEQ ID NOs 2, 5, 6, 7, 8, 9, 10, 11, 12, 23, 24, 26, positions 44 to 74 of SEQ ID NO 26, positions 77 to 105 of SEQ ID NO 26, and positions 89 to 105 of SEQ ID NO 26. The polypeptide can be synthetic, recombinant or a fusion protein. Polypeptide analogs of cortistatin are also described.
The invention also describes a substantially purified nucleic acid having a nucleotide sequence that encodes a cortistatin polypeptide having a sequence that corresponds to a sequence in the Sequence Listing selected from the group consisting of SEQ ID NOs 2, 5, 6, 7, 8, 9, 10, 11, 12, 23, 24, 26, positions 44 to 74 of SEQ ID NO 26, positions 77 to 105 of SEQ ID NO 26, and positions 89 to 105 of SEQ ID NO 26. The nucleic acid can be operatively linked to a promoter in an expression vector. Vectors for expressing cortistatin and cells containing the vectors are also described. Polynucleotide primers useful for hybridizing to cortistatin genes and gene products (e.g., mRNA) are also described.
The invention also contemplates an antibody that immunoreacts with cortistatin or with a polypeptide having a sequence that corresponds to a sequence in the Sequence Listing selected from the group consisting of SEQ ID NOs 2, 5, 6, 7, 8, 9, 10, 11, 12, 23, 24, 26, positions 44 to 74 of SEQ ID NO 26, positions 77 to 105 of SEQ ID NO 26, and positions 89 to 105 of SEQ ID NO 26. The antibody can also be a monoclonal antibody.
The invention also contemplates a kit for detecting the presence of cortistatin in a human body sample comprising an anti-cortistatin antibody, cortistatin polypeptide or oligonucleotide of the invention.
The invention further contemplates methods for detecting the presence of a nucleic acid that encodes cortistatin in a human body sample containing nucleic acid comprising the steps of:
(a) hybridizing the nucleic acid in the body sample with a oligonucleotide that includes at least 10 contiguous nucleotides from the nucleotide sequence shown in SEQ ID NO 1 from nucleotide 324 to nucleotide 366 to form a hybridization product; and
(b) detecting the presence of the hybridization product.
In a related method the invention describes a method of detecting the presence of a cortistatin antigen in a human body sample comprising the steps of:
(a) contacting a human body sample with an anti-cortistatin antibody that immunoreacts with human cortistatin or with a polypeptide having the amino acid residue sequence shown in SEQ ID NO 8 for a time period sufficient for said antibody to immunoreact with said antigen present in the sample and form an immunoreaction complex; and
(b) detecting the presence of an immunoreaction complex, thereby detecting said antigen.
Screening methods for identifying a ligand that binds to cortistatin receptor are also described which comprise:
(a) contacting a mammalian cell having a cortistatin receptor with a candidate ligand under conditions permitting binding of a known cortistatin receptor ligand to said cortistatin receptor; and
(b) detecting the presence of any of said candidate ligand bound to said receptor, or:
(a) contacting a mammalian cell having a cortistatin receptor with a candidate ligand under conditions permitting binding of a known cortistatin receptor ligand to said cortistatin receptor in the presence of a labeled cortistatin receptor ligand; and
(b) detecting the presence of any of said labeled ligand bound to said receptor.
Cortistatin polypeptides can also be used to directly detect the presence of a cortistatin receptor in a tissue sample comprising the steps of:
(a) contacting a tissue sample with an isolated cortistatin ligand under conditions permitting binding of a known cortistatin ligand to said cortistatin receptor; and
(b) detecting the presence of isolated cortistatin ligand bound to said tissue sample.
Therapeutic methods for altering cortistatin gene expression in a cell are contemplated comprising introducing into said cell an oligonucleotide capable of specifically hybridizing to the cortistatin gene. Alternatively, a method for activating the physiological response of cortistatin receptor upon binding to cortistatin is contemplated comprising contacting said cortistatin receptor with a pharmaceutical composition comprising a physiologically acceptable carrier and an effective activating amount of a cortistatin receptor agonist. Similarly, a cortistatin receptor antagonist can be used to inhibit the receptor.
Mutations in the cortistatin gene of a mammal that comprises an expansion of the CTG domain of the cortistatin gene can be assayed, comprising the steps of:
(a) determining the nucleotide sequence of the CTG domain of the cortistatin gene in a nucleic acid sample from said mammal; and
(b) comparing the determined nucleotide sequence to the known sequence of the CTG domain in a normal cortistatin gene to identify the presence of a sequence expansion in the CTG domain, and thereby said mutation.
The pharmacological activity of a cortistatin polypeptide can be exploited in a method for inducing sleep in a mammal comprising administering a physiologically tolerable composition containing a therapeutically effective amount of a cortistatin analog to said mammal. Similarly, sleep can be inhibited by use of a cortistatin receptor antagonist.
Other embodiments will be apparent to one skilled in the art.